Irradiation of red blood cells and anaerobic storage

ABSTRACT

A blood storage system comprising: a collection vessel for red blood cells; an oxygen or oxygen and carbon dioxide depletion device; a storage vessel for red blood cells; tubing connecting the collection vessel to the oxygen or oxygen and carbon dioxide depletion device and the oxygen or oxygen and carbon dioxide depletion device to the storage vessel; and a gamma or X-ray irradiating device is used to irradiate red blood cells stored in the vessel, storing red blood cells under anaerobic conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/289,722, filed Nov. 4, 2011, which is a non-provisionalapplication of U.S. Provisional Application 61/410,684, filed Nov. 5,2010. U.S. patent application Ser. No. 13/289,722, filed Nov. 4, 2011,is also a continuation-in-part of U.S. patent application Ser. No.12/901,350, filed Oct. 8, 2010, now U.S. Pat. No. 8,535,421, issued Sep.17, 2013, which is a non-provisional of U.S. Provisional Application No.61/331,693, filed May 5, 2010, the entireties of each of which areherein incorporated by reference.

BACKGROUND 1. Field

The present disclosure relates to a storage blood system having anoxygen/carbon dioxide depletion device and a blood storage bag for thelong-term storage of red blood cells (RBCs). More particularly, thepresent disclosure relates to a blood storage system that is capable ofremoving oxygen and carbon dioxide from the red blood cells prior tostorage and gamma and/or X-ray irradiating red blood cells either pre-or post-anaerobic treatment, as well as maintaining oxygen or oxygen andcarbon dioxide depleted states during storage, thereby prolonging thestorage life and minimizing deterioration of the deoxygenated red bloodcells.

2. Background of the Art

Adequate blood supply and the storage thereof is a problem facing everymajor hospital and health organization around the world. Often, theamount of blood supply in storage is considerably smaller than the needtherefore. This is especially true during crisis periods such as naturalcatastrophes, war and the like, when the blood supply is oftenperilously close to running out. It is at critical times such as thesethat the cry for more donations of fresh blood is often heard. However,unfortunately, even when there is no crisis period, the blood supply andthat kept in storage must be constantly monitored and replenished,because stored blood does not maintain its viability for long.

Stored blood undergoes steady deterioration which is, in part, caused byhemoglobin oxidation and degradation and adenosine triphosphate (ATP)and 2-3,biphosphoglycerate (DPG) depletion. Oxygen causes hemoglobin(Hb) carried by the red blood cells (RBCs) to convert to met-Hb, thebreakdown of which produces toxic products such as hemichrome, hemin andfree Fe³⁺. Together with the oxygen, these products catalyze theformation of hydroxyl radicals (OH.cndot.), and both the OH.cndot. andthe met-Hb breakdown products damage the red blood cell lipid membrane,the membrane skeleton, and the cell contents. As such, stored blood isconsidered unusable after 6 weeks, as determined by the relativeinability of the red blood cells to survive in the circulation of thetransfusion recipient. The depletion of DPG prevents adequate transportof oxygen to tissue thereby lowering the efficacy of transfusionimmediately after administration (levels of DPG recover once inrecipient after 8-48 hrs). In addition, these deleterious effects alsoresult in reduced overall efficacy and increased side effects oftransfusion therapy with stored blood before expiration date, when bloodolder than two weeks is used. Reduction in carbon dioxide content instored blood has the beneficial effect of elevating DPG levels in redblood cells.

There is, therefore, a need to be able to deplete oxygen and carbondioxide levels in red blood cells prior to storage on a long-term basiswithout the stored blood undergoing the harmful effects caused by theoxygen and hemoglobin interaction. Furthermore, there is a need to storeoxygen and carbon dioxide depleted red blood cells in bags containing orin a bag surrounded by a barrier film with oxygen and carbon dioxidedepletion materials. Furthermore, there is a need to optimize ATP andDPG levels in stored red blood cells by varying the depletion orscavenging constituents prior to and/or during storage depending uponthe needs of the recipient upon transfusion. Furthermore, the bloodstorage devices and methods must be simple, inexpensive and capable oflong-term storage of the blood supply.

Another issue relates to transfusion-associated graft-versus-hostdisease (TA-GVHD) which is a rare but nearly fatal complicationassociated with transfusion therapy in severely immuno-compromised bloodrecipients (for example, bone marrow transplant recipient, patientsreceiving aggressive chemotherapy, premature neonates). Prevention ofTA-GVHD requires complete removal of, or arrest of the proliferativepotential of T-lymphocytes from donor blood. Although leuko reductionfilters are widely in use, they are not adequate in prevention ofTA-GVHD because it cannot completely eliminate lymphocytes. Thus,lymphocyte inactivation by gamma-irradiation is currently the onlyrecommended method for TA-GVHD prevention. Since it is a nearly fatalside effect of transfusion, some hospitals and countries irradiate everyunit of RBC for TA-GVHD prevention. More commonly, RBC units ordered forspecific recipients are irradiated before dispensed to the bedside.

Accordingly, anaerobically stored RBC must be compatible with gamma- orX-ray irradiation treatment so that anaerobically stored blood can betransfused to patients requiring irradiated RBC.

Gamma-irradiation abrogates proliferation of T-lymphocytes by damagingthe DNA directly and via reactive oxygen species (ROS), namely hydroxylradicals produced during gamma-radiolysis of water. Although red bloodcells (RBC) do not contain DNA, ROS generated by gamma-irradiation havebeen shown to cause significant damage to the RBC. The major damageobserved includes: i) increased hemolysis; ii) increased K+ leak; iii)reduction in post-transfusion survival; and iv) reduced deformability.Such damage is similar to, but an exaggerated form of storage-induceddamage of RBC. The compromised status of RBC is well known to thephysicians who administer such compromised RBC. The FDA mandatesrestricted use of such RBC in terms of shortened shelf life aftergamma-irradiation (14 days) and/or 28 days total shelf life forirradiated units.

The irradiation of blood components has received increased attention dueto increasing categories of patients eligible to receive such blood toprevent transfusion-associated graft versus host disease. However,irradiation leads to enhancement of storage lesions, which could havedeleterious effects when such blood is transfused. It is well known inthe field that the main deleterious side-effect of radiation on RBC isoxidative damage caused by ROS.

Radiation damage to RBC in the presence of oxygen can occur in two ways;

-   -   i) By ROS generated during and immediately after irradiation.        ROS can reside in RBC lipid, then attack proteins and lipids in        vicinity later during storage, as well as to initiate        peroxidation cycle of lipid and protein using oxygen to fuel.    -   ii) Met-Hb and its denaturation products generated in i) above        act as catalysts to further cause ROS-mediated oxidative damage        during subsequent extended refrigerated storage of RBC. This is        an enhanced version of storage lesion development using O2.

On the other hand, there is ample literature suggesting ROS as a majorculprit in causing deterioration of red blood cell (RBC) duringrefrigerated storage at blood banks, and that storing RBC underanaerobic condition significantly reduce such damages. Studies haveshown that irradiated red blood cells that are oxygen and oxygen andcarbon dioxide depleted are equivalent or healthier (in terms of K+leakage, hemolysis and oxidized proteins/lipids) in comparison tonon-irradiated and non-oxygen and carbon dioxide depleted blood andnon-oxygen and carbon dioxide depleted irradiated blood. In the contextof the present application, the higher concentration of potassium in RBCstorage media was at levels that indicated red blood cell damage. Thepresent disclosure applies the finding of compatibility ofgamma-irradiation with anaerobically stored blood, as well as theprotective effects of anaerobic conditions in enhancing ATP, DPG and inreducing oxidative damage during refrigerated storage, to substantiallyreduce the negative or deleterious effect of gamma- and X-rayirradiation of RBCs in the presence of oxygen.

U.S. Pat. No. 5,362,442 to Kent describes adding a scavenger to bindfree radicals such as ethanol. U.S. Patent No. 61/875,572 to Platz etal. describes adding chemical sensitizers; U.S. Pat. No. 6,482,585 toDottori and U.S. Pat. No. 6,403,124, also to Dottori, describe addingL-carnitine or an alkanoul derivative to reduce RBC cell membrane damageinduced by irradiation. These additives are not required to prevent thedeleterious effects of irradiation on RBCs when treated anaeorobically.

SUMMARY

A method and system for gamma or X-ray irradiation of RBC underanaerobic or anaerobic and CO₂ depleted conditions, and extendedrefrigerated storage of such RBC under anaerobic or anaerobic and/or CO₂depleted conditions using an oxygen and/or CO₂ depletion device.

A method and system for removing plasma with or without platelets,adding an additive solution (e.g., nutrient and/or metabolicsupplements) to the concentrated RBC, filtering out leukocytes and/orplatelets via a leuko reduction filter, removing oxygen and/or CO₂ fromthe filtered RBC, and gamma irradiating or X-ray irradiating the oxygenand/or CO₂ filtered RBC either prior to or during storage thereof. Thepreferred range of gamma irradiation is a minimum of between about 25 Gyto 50 Gy.

Gamma or X-ray irradiating RBC under anaerobic or anaerobic and CO₂conditions (ambient to 1° C.) defined as less than 20% SO₂(oxygen-saturation of hemoglobin), more preferably less than 5%, andmost preferably less than 3%.

Storing gamma or X-ray irradiated (either under anaerobic or anaerobicand CO₂ conditions) RBC for extended time at 1-6° C. under anaerobiccondition defined as less than 20% SO₂ (oxygen-saturation ofhemoglobin), more preferably less than 5%, and most preferably less than3%.

Gamma or x-ray irradiating RBC under anaerobic or anaerobic and CO₂depleted conditions (ambient to 1° C.) defined as less than 20% SO₂(oxygen-saturation of hemoglobin), more preferably SO₂<5%, and mostpreferably SO₂<3% and pCO₂<10 mmHg; pCO₂<5 mmHg; pCO₂<1 mmHg.

Gamma or x-ray irradiating RBC under aerobic conditions (ambient to 1°C.) and then removing oxygen or oxygen and carbon dioxide from theirradiated RBC to levels defined as less than 20% SO₂ (oxygen-saturationof hemoglobin), more preferably SO₂<5%, and most preferably SO₂<3% andpCO₂<10 mmHg; pCO₂<5 mmHg; pCO₂<1 mmHg. The gamma or x-ray irradiationunder aerobic conditions and removal of oxygen or oxygen and carbondioxide can be performed before placing blood for extended storage, orwithin 24 hr of blood collection, between 1 through 7 days after bloodcollection or beyond 7 days

Using older blood, defined as blood stored for more than one week, andexposing such blood to gamma or x-ray irradiating RBC under aerobicconditions (ambient to 1° C.) and then removing oxygen or oxygen andcarbon dioxide from the irradiated RBC to levels defined as less than20% SO₂ (oxygen-saturation of hemoglobin), more preferably SO₂<5%, andmost preferably SO₂<3% and pCO₂<10 mmHg; pCO₂<5 mmHg; pCO₂<1 mmHg.

Using older blood, defined as blood stored for more than one week, andremoving oxygen or oxygen and carbon dioxide from such older blood andexposing such blood to Gamma or x-ray irradiation at wherein the levelsof oxygen and carbon dioxide are levels defined as less than 20% SO₂(oxygen-saturation of hemoglobin), more preferably SO₂<5%, and mostpreferably SO₂<3% and pCO₂<10 mmHg; pCO₂<5 mmHg; pCO₂<1 mmHg.

Storing gamma or X-ray irradiated or pre-irradiated RBC (either underanaerobic conditions with or without CO₂ depletion) RBC for extendedtime at 1-6° C. under anaerobic or anaerobic and CO₂ depleted conditiondefined as less than 20% SO₂ (oxygen-saturation of hemoglobin), morepreferably less than 5%, and most preferably 3% and less than pCO₂<10mmHg; pCO₂<5 mmHg; pCO₂<1 mmHg.

A preferred embodiment includes a blood storage system comprising: acollection vessel for red blood cells; an oxygen or oxygen/carbondioxide depletion device; tubing connecting the collection vessel to theoxygen or oxygen/carbon dioxide depletion device and the storage vesselfor red blood cells that can be gamma or X-ray irradiated and storedunder anaerobic or anaerobic and CO₂ depleted condition for extendedtime.

Preferably, the anaerobic or anaerobic and CO₂ condition is measured asan oxygen-saturation of hemoglobin of less than 20% SO₂, preferablyabout 5% or less, and most preferably about 3% or less.

The oxygen or oxygen/carbon dioxide depletion device comprises: acartridge; a plurality of gas permeable hollow fibers or sheetsextending within the cartridge from an entrance to an exit thereof,wherein the hollow fibers or gas-permeable films are adapted toreceiving and conveying red blood cells; and an amount of an oxygenscavenger or both oxygen scavenger and a carbon dioxide scavenger packedwithin the cartridge and contiguous to and in between the plurality ofhollow fibers.

Preferably, the oxygen or oxygen/carbon dioxide depletion devicecomprises: a cartridge; a plurality of hollow fibers or gas-permeablefilms extending within the cartridge from an entrance to an exitthereof, wherein the hollow fibers or gas-permeable films are adapted toreceiving and conveying red blood cells; and a low oxygen or a lowoxygen and carbon dioxide environment is created outside the hollowfibers by flowing an inert gas in-between the hollow fibers.

The blood storage system further comprising a leuko reduction filterdisposed between the collection vessel and the oxygen/carbon dioxidedepletion device. The blood storage system further comprising anadditive solution vessel in communication with the collection vessel.The blood storage system further comprising a plasma vessel incommunication with the collection vessel.

A method for storing red blood cells, the method comprising: removingoxygen or oxygen and carbon dioxide from red blood cells to produceanaerobic red blood cells; and storing irradiated RBC with either gamma-or X-ray, thereby producing irradiated anaerobic red blood cells; andstoring the irradiated anaerobic or anaerobic and CO₂ depleted red bloodcells.

The irradiated anaerobic or irradiated anaerobic and CO₂ depleted redblood cells are preferably stored at a temperature from between about 1°C. to about 6° C. under anaerobic conditions.

The present disclosure also provides for a device and method of removingcarbon dioxide (CO₂) in addition to oxygen (O₂) prior to or at the onsetof anaerobic or anaerobic and CO₂ depleted storage and/or gamma or X-rayirradiation.

The present disclosure provides for a blood collection system thatincorporates an oxygen or oxygen/carbon dioxide depletion device havingan oxygen or oxygen and carbon dioxide sorbent in combination with afilter or membrane to strip oxygen or oxygen and carbon dioxide from theblood during transport to the storage bag, wherein the oxygen/carbondioxide depleted blood is gamma or X-ray irradiated either prior to orduring storage.

The present disclosure further provides for a system to deplete theoxygen or oxygen and carbon dioxide from collected red blood cells thatincludes an (optional additive solution), an oxygen or oxygen and carbondioxide depletion device, and a blood storage bag that maintains the redblood cells in an oxygen or oxygen and carbon dioxide depleted stateafter gamma- or X-ray irradiation.

The present disclosure and its features and advantages will become moreapparent from the following detailed description with reference to theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a illustrates the components of a gamma irradiated, disposableblood anaerobic storage system of the present disclosure.

FIG. 1b illustrates the components of a second embodiment of a gammairradiated, disposable blood anaerobic storage system of the presentdisclosure.

FIG. 2a illustrates the components of an embodiment of a disposableblood anaerobic storage system that are used in conjunction with RBCirradiation in which red blood cells are irradiated during anaerobicstorage.

FIG. 2b illustrates the components of a second embodiment of adisposable blood anaerobic storage system that are used in conjunctionwith RBC irradiation.

FIG. 3 illustrates a pre-storage oxygen/carbon dioxide depletion deviceof the present disclosure.

FIG. 4 illustrates a first embodiment of a blood storage bag having astorage bag with a secondary outer oxygen film containing an oxygensorbent in a pocket.

FIG. 5a illustrates a pre-storage oxygen/carbon dioxide depletion baghaving a blood storage bag with a large sorbent sachet enclosed ingas-permeable, red blood cell compatible polymers in contact with theRBCs.

FIG. 5b illustrates a third embodiment of a blood storage bag having astorage bag a laminated oxygen film barrier with a large sorbent incontact with the RBCs.

FIG. 6a illustrates a fourth embodiment of a blood storage bag having asecondary configured secondary outer barrier bag surrounding an innerblood storage bag having an oxygen sorbent.

FIG. 6b illustrates a fifth embodiment of a blood storage bag having asecondary outer barrier bag surrounding an inner blood storage baghaving a large oxygen sorbent sachet enclosed in a gas permeable, redblood cell compatible polymers in contact with RBCs.

FIGS. 7a through 7c illustrate an embodiment of a depletion device thatdepletes oxygen and carbon dioxide from red blood cells prior to storageby a flushing inert gas or inert gas/CO₂ mixture of defined compositionaround a hollow fiber inside the assembly.

FIGS. 8a through 8c illustrate another embodiment of a depletion devicethat depletes oxygen and carbon dioxide from red blood cell prior tostorage.

FIGS. 9a through 9c illustrate another embodiment of a depletion devicethat depletes oxygen and carbon dioxide from red blood cells prior tostorage wherein oxygen and CO₂ is scavenged by scavenger materials inthe core of the cylinder, surrounded by hollow fibers.

FIGS. 10a through 10c illustrate another embodiment of a depletiondevice that depletes oxygen and carbon dioxide from red blood cellsprior to storage wherein oxygen and CO₂ is scavenged by scavengermaterials surrounding cylinders of hollow fibers enveloped in gaspermeable, low water vapor transmission material.

FIG. 11 illustrates a plot of flow rate of RBC suspension per minuteversus oxygen partial pressure for the depletion devices of FIGS. 7athrough 7c , FIGS. 8a through 8c , FIGS. 9a through 9c and FIGS. 10athrough 10 c.

FIGS. 12a through 12h illustrate plots of the effect of oxygen andoxygen and carbon dioxide depletion on metabolic status of red bloodcells during refrigerated storage.

FIG. 13 illustrates an effect of gamma-irradiation on K+ leak rates fromRBC (as measured by free K+ concentrations in RBC suspending media afterstorage).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

RBCs do not require oxygen for their own survival. It was shownpreviously that when RBCs were stored in blood bank refrigerator (1-6°C.) under anaerobic or anaerobic and CO₂ depleted conditions, theydemonstrated significantly improved post-transfusion recovery after6-week storage compared to the conventionally stored controls. Themechanisms of reduction in storage lesions under anaerobic oranaerobic/CO₂ depleted conditions have been described and directevidences demonstrated. It is, at least in part, due to reduction inoxidative damages in the presence of O₂ caused by ROS duringrefrigerated storage.

Because gamma- or X-ray irradiation exacerbate oxidative damage ontreated RBC, storing irradiated RBC under anaerobic and, optionally, CO₂depleted condition is not expected to intensify the damage; it is alsoexpected to prevent damage resulting from ROS generated duringirradiation by depriving O₂ that fuels those reactions.

Effectiveness of gamma- or X-ray irradiation is not dependent on thepresence of oxygen. In contrast, anaerobic condition is shown to be moreeffective in causing damage to DNA (and thus inhibiting proliferation oflymphocytes). Furthermore, absence of O₂ during and/or immediately aftergamma- or X-ray irradiation will reduce O₂-fueled oxidative damages toRBC induced by hydroxyl radicals and ROS produced by radiolysis of waterwith gamma- or X-rays.

Referring to the drawings and in particular to FIG. 1a , a disposableblood anaerobic storage system is shown and referenced using referencenumeral 10. The blood storage system includes an oxygen/carbon dioxidedepletion device 100 (OCDD 100), an anaerobic blood storage bag 200 andan additive solution bag 300. OCDD 100 removes oxygen and/or carbondioxide from red blood cells traveling through it. The system alsocontains a leuko reduction filter 400. Components conventionallyassociated with the process of blood collection are a phlebotomy needle410, a blood collection bag 420 containing an anti-coagulant and a bag430 containing plasma. Tubing can connect the various components of theblood storage system 10 in various configurations (one embodimentshown). Tube 440 connects collection bag 420 with leuko reduction filter400. Tube 441 connects additive solution bag 300 with collection bag420. Tube 442 connects plasma bag 430 with collection bag 420. Tube 443connects leukoreduction filter 400 with OCDD 100. Tube 414 connects OCDD100 with blood storage bag 200. Blood storage system 10 is preferably asingle-use, disposable, low cost system. As filtered and oxygen oroxygen and carbon dioxide depleted blood passes from OCDD 100 to bloodstorage bag 200. Blood stored in bag 200 will be gamma and/or X-rayirradiated during storage via device 453. Bag 200 containing oxygendepleted or oxygen and carbon dioxide depleted RBC is placed into device453 and exposed to gamma and/or X-ray radiation. Alternatively,pre-anaerobic blood stored in collection bag 421 can be gamma and/orX-ray irradiated via device 445 before passing through OCDD 100 andstored in bag 200, as shown in FIG. 1b . In FIG. 1b , bag 420 could alsobe gamma and/or X-ray irradiated in an irradiating device 445 prior topassing through leukoreduction filter 400.

Oxygen or oxygen/carbon dioxide depletion device 100 removes the oxygenfrom collected RBCs prior to the RBCs being stored in blood storage bag200. The oxygen content in RBCs must be depleted from oxy-hemoglobinbecause more than 99% of such oxygen is hemoglobin-bound in venousblood. Preferably, the degree of oxygen saturation is to be reduced toless than 4% within 48 hours of blood collection. The oxygen depletionis preferably accomplished at room temperature. The affinity of oxygento hemoglobin is highly dependent on the temperature, with a p50 of 26mmHg at 37° C. dropping to ˜4 mmHg at 4° C. Furthermore, this increasein O₂ affinity (Ka) is mainly due to reduction in O₂ release rate(k-off), resulting in an impractically low rate of oxygen removal onceRBC is cooled to 4° C. Thus, it places a constraint on oxygen strippingsuch that it may be preferable to accomplish it before RBC are cooled tostorage temperatures of 1° C. to 6° C.

In addition to oxygen depletion, carbon dioxide depletion has thebeneficial effect of elevating DPG levels in red blood cells. Carbondioxide exists inside RBCs and in plasma in equilibrium with HCO₃ ⁻ ion(carbonic acid). Carbon dioxide is mainly dissolved in RBC/plasmamixture as carbonic acid and rapid equilibrium between CO₂ and carbonicacid is maintained by carbonic anhydrase inside RBC. Carbon dioxide isfreely permeable through RBC membrane, while HCO₃ ⁻ inside RBC andplasma is rapidly equilibrated by anion exchanger (band 3) protein. WhenCO₂ is removed from RBC suspension, it results in the known alkalizationof RBC interior and suspending medium. This results from removal of HCO₃⁻ inside and outside RBC; cytosolic HCO₃ ⁻ is converted to CO₂ bycarbonic anhydrase and removed, while plasma HCO₃ ⁻ is removed via anionexchange inside RBC. Higher pH inside RBC is known to enhance the rateof glycolysis and thereby increasing ATP and DPG levels. ATP levels arehigher in Ar/CO₂ (p<0.0001). DPG was maintained beyond 2 weeks in theArgon purged arm only (p<0.0001). Enhanced glycolysis rate is alsopredicted by dis-inhibition of key glycolytic enzymes via metabolicmodulation and sequesterization of cytosolic-free DPG upon deoxygenationof hemoglobin as a result of anaerobic condition. DPG was lost at thesame rate in both control and Ar/CO₂ arms (p=0.6) despite thoroughdeoxygenation of hemoglobin, while very high levels of ATP were achievedwith OFAS3 additive (FIGS. 12a-12d ).

Referring to the drawings, and in particular to FIG. 2a , anotherembodiment of a disposable blood anaerobic storage system is shown andreferenced using reference numeral 500. The anaerobic conversion systemincludes an oxygen or oxygen/carbon dioxide depletion device 515 (OCDD)and an anaerobic blood storage bag 528. OCDD 515 removes oxygen oroxygen and carbon dioxide from red blood cells traveling through it.Tubing connects the various components of the blood storage system 500.Tube 512 connects to RBC concentrate prepared by using an additivesolution (e.g., AS1, AS3, AS5, SAGM, MAPS, etc.) and storing in bag 528by passing aforementioned RBC concentrate from collection bag 510through OCDD 515. Tubes 518 and 520 connect OCDD 515 with blood storagebag 528. Blood storage system 500 is preferably a single-use,disposable, low cost system. Oxygen and/or carbon dioxide depleted bloodis gamma and/or X-ray in blood storage bag 528 via device 553 andsubsequently stored for later transfusion.

Alternatively, blood in collection bag 510 may be gamma- or X-rayirradiated via device 551 prior to oxygen or oxygen and carbon dioxidedepletion and low temperature storage, as shown in FIG. 2b . FIG. 2bapplies to the scenario in which blood bag 510 contains older, forexample 2 day old blood, that is then irradiated and depleted of oxygenor oxygen and or carbon dioxide, and stored.

Referring to FIG. 3, an oxygen or oxygen/carbon dioxide depletion device(OCDD) 101 contains an oxygen sorbent 110. OCDD 101 is a disposablecartridge 105 containing oxygen sorbent 110 and a series of hollowfibers 115. Oxygen sorbent 110 is a mixture of non-toxic inorganicand/or organic salts and ferrous iron or other materials with highreactivity toward oxygen. Oxygen sorbent 110 is made from particles thathave significant absorbing capacity for O₂ (more than 5 ml O₂/g) and canmaintain the inside of cartridge 105 to less than 0.01% whichcorresponds to PO₂ less than 0.08 mmHg. Oxygen sorbent 110 is eitherfree or contained in an oxygen permeable envelope. OCDD 101 of thepresent disclosure must deplete approximately 100 mL of oxygen from aunit of blood.

After oxygen and, optionally, carbon dioxide have been stripped fromRBCs in the OCDD of FIG. 3, RBCs are stored in a blood storage bag 200.The oxygen content of RBC suspended in additive solution 300 must bereduced to equal to or less than 4% SO₂ before placing them inrefrigerated storage. Further, oxygen depleted RBC must be kept in ananaerobic state and low carbon dioxide state throughout entire storageduration.

RBCs pass through an oxygen permeable film or membrane, that may beformed as hollow fibers 115 of FIG. 3. The membrane or films may beconstructed in a flat sheet or hollow fiber form. The oxygen permeablefilms can be non porous materials that are capable of high oxygenpermeability rates (polyolefins, silicones, epoxies, polyesters, etc.)and oxygen permeable membranes are hydrophobic porous structures. Thesemay be constructed of polymers (e.g., polyolefins, Teflon, PVDF, orpolysulfone) or inorganic materials (e.g., ceramics). Oxygen depletiontakes place as RBC pass through hollow fibers 115. Oxygen permeablefilms or oxygen permeable membranes may be extruded into sheets orhollow fibers 15. Accordingly, hollow fibers 115 and sheets may be usedinterchangeably. OCDD provides a simple structure having a large surfacearea to remove oxygen and maintain constant flow of blood therethrough.The oxygen depletion or removal is accomplished by irreversible reactionof ferrous ion in oxygen sorbent 110 with ambient oxygen to form ferricoxide. OCDD 101 does not need agitation for oxygen removal and can bemanufactured easily to withstand centrifugation as part of a bloodcollection system as necessary.

Referring to FIGS. 7a through 7c and FIGS. 8a through 8c , examples offlushing depletion devices are disclosed. The depletion devices functionto deplete, O₂ and CO₂, or O₂ alone, or O₂ with specific levels of CO₂by supplying appropriate composition of flushing gas. Gases appropriatefor depletion devices are, for example, Ar, He, N₂, Ar/CO₂, or N₂/CO₂.

FIGS. 9a through 9c and 10a through 910c , also disclose scavengingdepletion devices. Depletion takes place with the use of scavengers orsorbents and without the use of external gases. In both types ofdepletion devices however, carbon dioxide depletion in conjunction withoxygen depletion is effective to enhance DPG and ATP, respectively,prior to storage in blood storage bags.

Referring to FIGS. 7a through 7c , a depletion device 20 is shown.Depletion device 20 includes a plurality of fibers 25, approximately5000 in number, through which red blood cells flow. Plurality of fibers25 are surrounded by a plastic cylinder 30. Plastic cylinder orcartridge 30 contains a gas inlet 35 and a gas outlet 40 through which aflushing gas or a combination of flushing gases, such as those mentionedabove, are supplied to remove carbon and/or oxygen from blood.Specifications for depletion device 20 are shown in Table 1 below atsecond column.

TABLE 1 External Gas Externa Gas Prototype Specification PathwaysPathways Prototype Serial #: Device 20 Device 45 Fiber Type: CelgardCelgard 200/150-66FPI 200/150-66FPI Number of Fibers: 5000 5000 ActiveLength of Fibers (cm): 13 28 Fiber OD (microns): 200 200 Fiber ID(microns): 150 150 Total Length of Fibers 15 30 Active Fiber SurfaceArea (m2): 0.4084 0.8796

Referring to FIGS. 8a through 8c , a depletion device 45 is shown.Depletion device 45, like device 20 of FIGS. 7a to 7c , includes aplurality of fibers 50, approximately 5000 in number, through which redblood cells flow. Plurality of fibers 50 are surrounded by a plasticcylinder 55. Plastic cylinder 55 contains a gas inlet 60 and a gasoutlet 65 through which a gas or a combination of gases, such as thosementioned above are supplied to remove oxygen or oxygen and carbondioxide from blood. Specifications for depletion device 45 are shown inTable 1 above in the third column. The active surface area of depletionof device 45 is twice that of device 20 because device 45 is twice aslong as device 20.

FIGS. 9a through 9c disclose a depletion device 70 having a core 75containing scavenging materials for either O₂, or both O₂ and CO₂. Core75 is packed by a gas permeable film with very low liquid permeability.Hollow fibers 80 are wound around core 75, and a plastic cylinder 82contains and envelopes hollow fibers 80. In this particular embodiment,the active surface area for depletion is approximately 0.8796 m² asshown in Table 2 below at the second column.

TABLE 2 Center Core 10 individual 125 grams Bundles 200 grams PrototypeSpecification Sorbent Sorbent Prototype Serial #: Device 70 Device 85Fiber Type: Celgard Celgard 200/150-66FPI 200/150-66FPI Number ofFibers: 5000 5000 Active Length of Fibers (cm): 13 28 Fiber OD(microns): 200 200 Fiber ID (microns): 150 150 Total Length of Fibers 1530 Active Fiber Surface Area (m2): 0.8796 0.8796

FIGS. 10a through 10c disclose a depletion device 85 containing fiberbundles 87 enclosed in gas permeable film with very low liquidpermeability. Fiber bundles 87 are surrounded by scavenger materials 89for either O₂ or both O₂ and CO₂. Fiber bundles 87 and scavengermaterials 89 are contained within a plastic cylinder 90. The activesurface area for depletion is approximately 0.8796 m² as shown in Table2 above at the third column.

FIG. 11 is a plot of the performance of flushing depletion devices 20and 45 and scavenging depletion devices 70 and 85. The data of FIG. 11was plotted using the following conditions: Hematocrit, 62% (pooled 3units of pRBC), and 21° C. at various head heights to produce differentflow rates. Oxygen/carbon dioxide scavenger (Multisorb Technologies,Buffalo, N.Y.) was activated with adding 5% and 12% w/w water vapor fordevice 79 and device 85, respectively. Data are plotted with flow rate(g RBC suspension per min) vs. pO₂ (mmHg).

In the oxygen/carbon dioxide depletion devices disclosed herein, aplurality of gas permeable films/membranes may be substituted for theplurality of hollow fibers. The films and fibers may be packed in anysuitable configuration within the cartridge, such as linear orlongitudinal, spiral, or coil, so long as they can receive and conveyred blood cells.

FIG. 11 shows that lowest oxygen saturation is achieved using devices 45and 85. Device 45 exhibits a larger active surface area exposed to gasesalong length of fibers 50. Device 85 also has a long surface area ofexposure to scavenging materials. Device 85 has bundles 87 surrounded byscavenging materials 89. The space occupied by scavenging materials 89between bundles 87 promotes dispersion of oxygen and carbon dioxide fromred blood cells contained in fiber bundles 87, thus aiding scavenging ofoxygen and carbon dioxide from red blood cells.

A further use of the depletion devices is to add back oxygen and orcarbon dioxide prior to transfusion by flushing with pure oxygen or air.This use is for special cases, such as massive transfusions, where thecapacity of the lung to re-oxygenate transfused blood is not adequate,or sickle cell anemia.

Similarly, depletion devices can be used to obtain intermediate levelsor states of depletion of oxygen and carbon dioxide depending needs ofthe patient to obtain optimal levels in the transfused blood dependingupon the patients needs.

Referring to FIG. 4, a blood storage bag 200 according to a preferredembodiment of the present disclosure is provided. Blood bag 200 has aninner blood-compatible bag 250 (preferably polyvinyl chloride (PVC)),and an outer barrier film bag 255. The material of bag 250 is compatiblewith RBCs. Disposed between inner bag 250 and outer oxygen barrier filmbag 255 is a pocket that contains an oxygen/carbon dioxide sorbent 110.Barrier film bag 255 is laminated to the entire surface of inner bag250. Sorbent 110 is contained in a sachet 260, which is alternatelyreferred to as a pouch or pocket. Sorbent 110 is optimally locatedbetween tubing 440 that leads into and from bag 200, specificallybetween inner bag and outer oxygen barrier film bag 255. This locationwill ensure that oxygen disposed between these two bags will bescavenged or absorbed. Oxygen sorbent is ideally located in a pouch orpocket 260 and not in contact with RBCs. Oxygen sorbent may also becombined with CO₂ scavengers or sorbents, enabling sorbent 110 todeplete both oxygen and carbon dioxide at the same time.

Referring to FIGS. 5a and 5b , blood storage bags 201 and 202 areconfigured to store RBCs for extended storage periods of time. Innerblood storage bags 205 are preferably made from DEHP-plasticized PVC andare in contact with RBCs. DEHP-plasticized PVC is approximately 200 foldless permeable to oxygen compared to silicone. However, PVC isinsufficient as an oxygen barrier to maintain the anaerobic state ofRBCs throughout the storage duration. Therefore, blood storage bags 201and 202 are fabricated with outer transparent oxygen barrier film 206(e.g., nylon polymer) laminated to the outer surface inner blood bag205. This approach, as well as one shown in FIG. 3, uses accepted PVCfor blood contact surface (supplying DEHP for cell stabilization) at thesame time prevents oxygen entry into the bag during extended storage.

In FIG. 5a , a small sachet 210 containing oxygen/carbon dioxide sorbent110 enveloped in oxygen-permeable, RBC compatible membrane is enclosedinside of laminated PVC bag 205 and in contact with RBCs. Small sachetenvelope 210 is preferably made from a silicone or siloxane materialwith high oxygen permeability of biocompatible material. Sachet envelope210 has a wall thickness of less than 0.13 mm thickness ensures that O₂permeability ceases to become the rate-limiting step. PVC bag 205 mayalso contain carbon dioxide scavengers.

Referring to FIG. 5b , bag 202 has a similar configuration to bag 201 ofFIG. 4a . However, bag 202 has a large sorbent 215 enclosed inside ofPVC bag 205. Large sorbent 215 preferably has a comb-like configurationto rapidly absorb oxygen during extended storage. The benefit oflaminated bags of FIGS. 4a and 4b is that once RBCs are anaerobicallystored in bags, no further special handling is required. Similarly, bag202 may contain carbon dioxide scavenger to provide carbondioxide-scavenging in addition to oxygen-scavenging capability.

Referring to the embodiments of FIGS. 6a and 6b , RBCs are stored insecondary bags 301 and 302, respectively, in order to maintain ananaerobic storage environment for RBC storage. Secondary bags 301 and302 are transparent oxygen barrier films (e.g., nylon polymer) thatcompensate for the inability of PVC blood bags 305 and 320,respectively, to operate as a sufficient oxygen barrier to maintain RBCsin an anaerobic state. Secondary bags 301 and 302 are made with anoxygen barrier film, preferably a nylon polymer or other transparent,flexible film with low oxygen permeability.

Referring to FIG. 6a , a small oxygen/carbon dioxide sorbent 310 isdisposed between a PVC barrier bag 305 and secondary bag 306 to removeslowly diffusing oxygen. FIG. 6a is similar to the preferred embodimentof the blood bag of FIG. 4 except that secondary bag 306 is separatefrom and not bonded to bag 305 in this embodiment. PVC bag 305 includingports are enclosed in secondary barrier bag 305. Oxygen sorbent 310 mayoptionally contain carbon dioxide scavengers to provide both oxygen andcarbon dioxide scavenging capability.

Referring to FIG. 6b , a secondary bag 302 contains a large sachet 325inside of PVC bag 320. Sachet 325 is filled with either oxygen oroxygen/carbon dioxide sorbent 110. Sachet 325 is a molded element withsurface texture to increase the surface area. Sachet 325 has a comb-likegeometry for rapid oxygen or oxygen/carbon dioxide depletion. Sachet 325acts rapidly to strip oxygen or oxygen/carbon dioxide from RBCs prior torefrigeration and storage of RBCs in place of OCDD of FIG. 3. However,with this configuration, agitation is necessary, therefore sachet 325must possess a large surface area, high oxygen or oxygen/carbon dioxidepermeability and mechanical strength to withstand centrifugation stepduring component preparation and the prolonged storage. Sachet 325 ispreferably made from materials such as 0.15 mm thick silicone membranewith surface texture to increase the surface area. Sachet 325 may bemade from materials such as PTFE or other fluoropolymer. Sachet 325 mayhave a rectangular shape such, such as, for example, a 4″×6″ rectangle,although other sizes are possible, for the anaerobic maintenance. Sachet325 may contain carbon dioxide scavengers in addition to oxygenscavengers to provide oxygen and carbon dioxide scavenging capability.

The embodiments of FIGS. 6a and 6b are easily made from off-shelfcomponents except for sachet 325 of FIG. 6b . In order to access RBCsfor any testing, secondary bags 301 and 302 must be opened. Unless theunit is transfused within short time, RBC must be re-sealed with freshsorbent for further storage. (1 day air exposure of storage bag wouldnot oxygenate blood to appreciable degree, since PVC plasticized withDEHP has relatively low permeability to oxygen).

In FIGS. 5a, 5b, 6a and 6b , the PVC bag is preferably formed with theoxygen barrier film, such as a SiO₂ layer formed with the sol-gelmethod. A portion of the sheet material will be sealed on standard heatsealing equipment, such as radiofrequency sealers. Materials options maybe obtained in extruded sheets and each tested for oxygen barrier,lamination integrity, and seal strength/integrity.

For each of the several embodiments addressed above, an additivesolution from bag 300 is provided prior to stripping oxygen and carbondioxide from the RBCs is used. The additive solution 300 preferablycontains the following composition adenine 2 mmol/L; glucose 110 mmol/L;mannitol 55 mmol/L; NaCl 26 mmol/L; Na₂HPO₄ 12 mmol/L citric acid and apH of 6.5. Additive solution 300 is preferably an acidic additivesolution OFAS3, although other similar additive solutions could also beused that are shown to enhance oxygen/carbon dioxide-depleted storage.OFAS3 has shown enhanced ATP levels and good in vivo recovery asdisclosed herein. While OFAS3 is a preferred additive solution, othersolutions that offer similar functionality could also be used.Alternatively, additive solutions used currently in the field, such asAS1, AS3, AS5, SAGM, and MAPS can also be used. Additive solutions helpto prevent rapid deterioration of RBCs during storage and are typicallyadded prior to RBCs being made anaerobic.

Additionally, we envision that the OCDD and storage bags 100 and 200 canbe manufactured independent of other components of the disposable,anaerobic blood storage system (i.e., every item upstream of andincluding leuko reduction filter 400 in FIG. 1a ).

It is within the scope of the present disclosure to remove oxygen fromthe RBCs or to strip oxygen and carbon dioxide from the blood prior tostorage in the storage bags. An oxygen scavenger can be used to removethe oxygen from the RBCs prior to storage in the blood bags. As usedherein, “oxygen scavenger” is a material that irreversibly binds to orcombines with oxygen under the conditions of use. For example, theoxygen can chemically react with some component of the material and beconverted into another compound. Any material where the off-rate ofbound oxygen is zero can serve as an oxygen scavenger. Examples ofoxygen scavengers include iron powders and organic compounds. The term“oxygen sorbent” may be used interchangeably herein with oxygenscavenger. As used herein, “carbon dioxide scavenger” is a material thatirreversibly binds to or combines with carbon dioxide under theconditions of use. For example, the carbon dioxide can chemically reactwith some component of the material and be converted into anothercompound. Any material where the off-rate of bound carbon dioxide iszero can serve as a carbon dioxide scavenger. The term “carbon dioxidesorbent” may be used interchangeably herein with carbon dioxidescavenger. For example, oxygen scavengers and carbon dioxide scavengersare provided by Multisorb Technologies (Buffalo, N.Y.) or Mitsubishi GasChemical Co (Tokyo, Japan). Oxygen scavengers may exhibit a secondaryfunctionality of carbon dioxide scavenging. Such materials can beblended to a desired ratio to achieve desired results.

Carbon dioxide scavengers include metal oxides and metal hydroxides.Metal oxides react with water to produce metal hydroxides. The metalhydroxide reacts with carbon dioxide to form water and a metalcarbonate. For example, if calcium oxide is used, the calcium oxide willreact with water that is added to the sorbent to produce calciumhydroxideCaO+H₂O→Ca(OH)₂

The calcium hydroxide will react with carbon dioxide to form calciumcarbonate and water.Ca(OH)₂+CO₂→CaCO₃+H₂O

It will be appreciated that scavengers can be incorporated into storagereceptacles and bags in any known form, such as in sachets, patches,coatings, pockets, and packets.

If oxygen removal is completed prior to introduction of the RBCs to theblood storage device, then it can be accomplished by any method known inthe art. For example, a suspension of RBCs can be repeatedly flushedwith an inert gas (with or without a defined concentration of carbondioxide), with or without gentle mixing, until the desired oxygen and orcarbon dioxide content is reached or until substantially all of theoxygen and carbon dioxide has been removed. The inert gas can be argon,helium, nitrogen, mixtures thereof, or any other gas that does not bindto the hememoiety of hemoglobin.

The OCDDs and various storage bags of the present disclosure can be usedin varying combinations. For example, OCDD 101 of FIG. 3 can be usedwith blood bag of FIG. 4, 201 of FIG. 5a or 301 of FIG. 6a . When oxygenis depleted by in-bag sachet 215 of FIG. 6b , it can be stored as inFIG. 6b or oxygen/carbon dioxide-depleted content transferred to thefinal storage bag such as FIG. 4, FIG. 5a or FIG. 6a for extendedstorage. Other combinations and configurations are fully within thescope of the present disclosure.

The present disclosure also provides another embodiment of a bloodstorage device. The device is a sealed receptacle adapted to retain andstore red blood cells. The receptacle has walls formed from a laminate.The laminate has (a) an outer layer of a material substantiallyimpermeable to oxygen or oxygen and carbon dioxide, (b) an inner layerof a material compatible with red blood cells, and (c) an interstitiallayer between the outer layer and the inner layer. The interstitiallayer is of a material having admixed therein an amount of an oxygenscavenger or an oxygen/carbon dioxide scavenger. The layers preferablytake the form of polymers. A preferred polymer for the outer layer isnylon. A preferred polymer for inner layer is PVC. The polymer of theinterstitial layer should provide effective adhesion between the innerand outer layers and provide effective admixture of oxygen scavengers oroxygen/carbon dioxide scavengers therein. Useful polymers for theinterstitial layer include, for example, olefin polymers, such asethylene and propylene homopolymers and copolymers, and acrylicpolymers.

The present disclosure also provides another embodiment of a bloodstorage system. The system has a collection bag for red blood cells; aunitary device for depleting oxygen or oxygen and carbon dioxide andreducing leukocytes and/or platelets from red blood cells; a storage bagfor red blood cells; and tubing connecting the collection bag to theunitary device and the unitary device to the storage bag. A feature ofthis embodiment is that the functions of depleting oxygen or oxygen andcarbon dioxide and reducing leukocytes and/or platelets from red bloodcells are combined into a single, unitary device rather than requireseparate devices. For instance, unitary device can take the form of asingle cartridge. Leukocyte and/or platelet reduction is typicallycarried out by passing red blood cells through a mesh. In thisembodiment, a mesh can be incorporated into either the flushing or thescavenging oxygen or oxygen/carbon dioxide depletion device disclosedherein. The mesh is preferably located within the device so thatleukocyte and/or platelet reduction takes place prior to the onset offlushing or scavenging.

The following are examples of the present disclosure and are not to beconstrued as limiting.

EXAMPLES

FIGS. 12a through 12h show the results of a 3-arm study showing: acontrol (aerobic OFAS3 with no O₂ or CO₂ depletion), anaerobic OFAS3(both O₂ and CO₂ depleted with pure Ar), and O₂ only depleted with 95%Ar and 5% CO₂ (CO₂ is not depleted).

Whole blood was collected into CP2D (Pall), centrifuged 2K×G for 3minutes, plasma removed, and additive solution AS-3 (Nutricel, Pall), orexperimental OFAS3 added. The unit was evenly divided into 3 600 mLbags. 2 bags were gas exchanged ×7 with Ar or Ar/CO₂, transferred to 150mL PVC bags and stored 1° C. to 6° C. in anaerobic cylinders with Ar/H₂or Ar/H₂/CO₂. One control bag was treated in the same manner without agas exchange and stored 1° C. to 6° C. in ambient air. Bags were sampledweekly for up to 9 weeks.

The plots of FIGS. 12a, 12c, 12e and 12g : use the additive solutionOFAS3 (200 mL; experimental, proprietary) and the plots of FIGS. 12b,12d, 12f and 12h , use the AS-3 additive solution. Comparing additivesolutions, effects of CO₂ depletion on DPG levels were similar. OFAS3showed higher ATP when oxygen was depleted (±CO₂), and O₂ depletionalone showed significant enhancement of ATP compared to aerobic control.AS-3 additive exhibited no significant enhancement of ATP when O₂ alonewas depleted.

FIGS. 12a and 12b : DPG levels during storage. DPG levels weremaintained for over 2 weeks, when CO₂ was removed in addition to oxygen.

FIG. 12c : ATP levels during storage with OFAS3. Highest ATP levels wereachieved with OFAS3 RBC when O₂ only was depleted. For O₂/CO₂ depletion,intermediate levels of ATP were observed compared to the control whilevery high DPG levels were attained during first 2.5 weeks. Very highlevels of ATP may suggest higher rate of 24-hour post transfusionrecovery. Therefore, extent of carbon dioxide and oxygen depletionlevels may be adjusted to meet the specific requirement of therecipient. DPG levels can be maintained very high (at the expense ofATP) for purposes of meeting acute oxygen demand of recipient.Conversely, very high ATP levels may allow higher 24-hour recovery rate(lower fraction of non-viable RBC upon transfusion) thereby reducing thequantity of blood needed to be transfused (up to 25% of RBC arenon-viable). More importantly, this would benefit chronically transfusedpatients who may not demand highest oxygen transport efficiencyimmediately after transfusion (DPG level recovers in body after 8-48hours) who suffers from toxic iron overloading caused by non-viableRBCs.

FIG. 12d : ATP levels during storage with AS3. Highest ATP levels wereachieved with AS3 RBC when O₂ only was depleted. No significantdifferences in ATP levels where observed with control and O₂ depletionalone.

FIGS. 12e and 12f : pH of RBC cytosol (in) and suspending medium (ex).Immediately after gas exchange (day 0), significant rise in pH (in andex) was observed only when CO₂ was depleted together with O₂. Rapidrates of pH decline observed with CO₂/O₂ depleted samples were caused byhigher rates of lactate production (FIGS. 12g and 12h ).

FIGS. 12g and 12h : Normalized (to hemoglobin) glucose and lactatelevels during storage with OFAS3 and AS3. Higher rates of glucosedepletion and lactate productions correspond to high DPG levels observedin panels A and B. Legends for symbols/lines are same for both panels.OFAS3 additive contains similar glucose concentration with ×2 volumeresulting in higher normalized glucose levels.

FIGS. 12a and 12c taken together, suggest that extent of increases(compared to control) of ATP and DPG levels may be adjusted bycontrolling level of CO₂ depletion, when O₂ is depleted. Higher glucoseutilization and lactate production were observed with enhanced DPGproduction (FIG. 12g ). This may be also effective with AS3 additive,since similar trend in glucose utilization and lactate production wereobserved (FIG. 12h ).

FIG. 13 shows a graph comparing the effect of gamma irradiation onaerobic and anaerobic RBC. FIG. 13 shows an control unit, RBC that areaerobic and not gamma-irradiated (Unit A, black filled solid line),aerobic RBC that are gamma-irradiated (Unit B; control plus gammairradiation indicated by a filled circle with dotted line) and ananaerobically depleted RBC unit that has been gamma-irradiated (Unit C;Anaerobic+γ, open circle and solid line). Unit B and Unit C areirradiated and Unit A is non-irradiated and aerobic RBC. The constituentof the blood that is being measured is potassium. The amount of leakageof potassium (K+) from RBC that is measured in the storage media is anindicator of health of the RBC. Therefore, in the context of the presentapplication, a greater level of concentration of potassium in RBCstorage media, is indicative of a greater level of RBC damage relativeto a lower level of concentration of potassium in RBC storage media.

FIG. 13 indicates that gamma irradiation induced a high rate of K+leakage during the first week for Unit B and Unit C. K+ leakage ratesafter days eight and fifteen, were similar for all units. Significantly,the difference between K+ leakage between Unit B and Unit C increasesbeyond the twenty-second day of storage. The results indicate that thistrend could exist for several more days. Accordingly, the use ofanaerobic depletion and gamma irradiation may permit the extension ofcurrent FDA storage limit of twenty-eight days for anaerobicallydepleted and gamma irradiated blood prepared after component separation.

Irradiating RBC for immuno-compromised individuals is a necessity. Thepresent results show that irradiated RBC that were also oxygen depleteddid not increase K+ leakage rates, an indicator of RBC damage. Thebenefits of oxygen depleted RBC including increased levels of ATP andDPG-2,3 are not negatively impacted by the irradiation.

In graph above, four ABO Rh identical units (in AS3 additive,leukoreduced; standard RBC concentrate obtained from American Red Cross)are pooled. The three units were used for above-graphed experiment fromthe pooled unit after it was sub-divided into 4 fractions within 24hours of blood collection and stored at 1-6° C.

Although the present disclosure describes in detail certain embodiments,it is understood that variations and modifications known to thoseskilled in the art that are within the disclosure. Accordingly, thepresent disclosure is intended to encompass all such alternatives,modifications and variations that are within the scope of the disclosureas set forth in the disclosure.

What is claimed is:
 1. A composition comprising gamma-irradiated, oxygenand carbon dioxide-depleted red blood cells without added L-carnitine oran alkanoyl derivative, wherein said gamma-irradiated, oxygen and carbondioxide-depleted red blood cells are prepared by storing oxygen andcarbon dioxide-depleted red blood cells for a period of time and thengamma-irradiating to form said gamma-irradiated, oxygen and carbondioxide-depleted red blood cells, wherein said gamma-irradiated, oxygenand carbon dioxide-depleted red blood cells exhibit reduced potassiumleakage compared to red blood cells having been conventionally storedfor a period of at least 22 days.
 2. The composition of claim 1, whereinsaid composition comprises reduced lesions compared to a compositioncomprising red blood cells conventionally stored for an equivalentperiod of time and then gamma-irradiated.
 3. The composition of claim 1,wherein said composition comprises reduced oxidative damage compared toa composition comprising red blood cells conventionally stored for anequivalent period of time and then gamma-irradiated.
 4. The compositionof claim 1, wherein said gamma-irradiated oxygen and carbon dioxidedepleted red blood cells comprise less than 20% oxygen-saturation ofhemoglobin (SO₂).
 5. The composition of claim 1, wherein saidgamma-irradiated oxygen and carbon dioxide depleted red blood cellscomprise reduced irradiation damage compared to red blood cellsconventionally stored for an equivalent period of time and thengamma-irradiated.
 6. The composition of claim 1, wherein saidgamma-irradiated oxygen and carbon dioxide depleted red blood cellscomprises a partial pressure of carbon dioxide (pCO2) of less than 10millimeter of mercury (mmHg).
 7. The composition of claim 1, whereinsaid gamma-irradiated oxygen and carbon dioxide depleted red blood cellscomprise increased deformability compared to red blood cellsconventionally stored for an equivalent period of time and thenirradiated.
 8. The composition of claim 1, wherein said gamma-irradiatedoxygen and carbon dioxide depleted red blood cells comprise reducedhemolysis compared to red blood cells conventionally stored for anequivalent period of time and then irradiated.
 9. The composition ofclaim 1, wherein said gamma-irradiated oxygen and carbon dioxidedepleted red blood cells exhibit improved post-transfusion recoverycompared to red blood cells conventionally stored for an equivalentperiod of time and then gamma-irradiated.
 10. The composition of claim9, wherein said improved post-transfusion recovery is after 6 weeks ofstorage.
 11. The composition of claim 1, wherein said compositionfurther comprises an additive solution.
 12. The composition of claim 11,wherein said additive solution is AS-3 or OFAS3.
 13. The composition ofclaim 1, wherein said composition is leukoreduced.
 14. The compositionof claim 1, wherein said composition is free of platelets.
 15. Thecomposition of claim 1, wherein said composition is stored within astorage vessel substantially impermeable to oxygen or both oxygen andcarbon dioxide.
 16. The composition of claim 15, wherein said storagevessel comprises an inner layer compatible with blood and an outer layersubstantially impermeable to oxygen or both oxygen and carbon dioxide.17. A method of treating a patient at risk of developingtransfusion-associated graft-versus-host disease (TA-GVHD), comprisingproviding a composition to a patient, wherein said composition comprisesgamma-irradiated, oxygen and carbon dioxide-depleted red blood cellswithout added L-carnitine or an alkanoyl derivative, wherein saidgamma-irradiated, oxygen and carbon dioxide-depleted red blood cells areprepared by storing oxygen and carbon dioxide-depleted red blood cellsfor a period of time and then gamma-irradiating to form saidgamma-irradiated, oxygen and carbon dioxide depleted red blood cells,wherein said gamma-irradiated, oxygen and carbon dioxide-depleted redblood cells exhibit reduced potassium leakage compared to red bloodcells having been conventionally stored for a period of at least 22days, wherein said method reduces a risk of TA-GVHD in said patient. 18.The method of claim 17, wherein said patient is a patient receivingchemotherapy.
 19. The method of claim 17, wherein said patient isimmuno-compromised.
 20. The composition of claim 1, wherein saidgamma-irradiated oxygen and carbon dioxide depleted red blood cellscomprise less than 3% oxygen-saturation of hemoglobin (SO₂).